A. Field of the Invention
This invention relates to the field of methods and devices for performing nucleic acid amplification reactions.
B. Description of Related Art
Nucleic acid based amplification reactions are now widely used in research and clinical laboratories for the detection of genetic and infectious diseases. The currently known amplification schemes can be broadly grouped into two classes, based on whether, after an initial denaturing step (typically performed at a temperature of xe2x89xa765 degrees C.) for DNA amplifications or for RNA amplifications involving a high amount of initial secondary structure, the reactions are driven via a continuous cycling of the temperature between the denaturation temperature and a primer annealing and amplicon synthesis (or polymerase activity) temperature (xe2x80x9ccycling reactionsxe2x80x9d), or whether the temperature is kept constant throughout the enzymatic amplification process (xe2x80x9cisothermal reactionsxe2x80x9d). Typical cycling reactions are the Polymerase and Ligase Chain Reaction (PCR and LCR, respectively). Representative isothermal reaction schemes are NASBA (Nucleic Acid Sequence Based Amplification), Transcription Mediated Amplification (TMA), and Strand Displacement Amplification (SDA). In the isothermal reactions, after the initial denaturation step (if required), the reaction occurs at a constant temperature, typically a lower temperature at which the enzymatic amplification reaction is optimized.
Prior to the discovery of thermostable enzymes, methodologies that used temperature cycling were seriously hampered by the need for dispensing fresh polymerase into an amplification tube (such as a test tube) after each denaturation cycle, since the elevated temperature required for denaturation inactivated the polymerase during each cycle. A considerable simplification of the PCR assay procedure was achieved with the discovery of the thermostable Taq polymerase (from Thermophilus aquaticus). This improvement eliminated the need to open amplification tubes after each amplification cycle to add fresh enzyme. This led to the reduction of both the contamination risk and the enzyme-related costs. The introduction of thermostable enzymes has also allowed the relatively simple automation of the PCR technique. Furthermore, this new enzyme allowed for the implementation of simple disposable devices (such as a single tube) for use with temperature cycling equipment.
TMA requires the combined activities of at least two (2) enzymes for which no optimal thermostable variants have been described. For optimal primer annealing in the TMA reaction, an initial denaturation step (at a temperature of xe2x89xa765 degrees C.) is performed to remove secondary structure of the target. The reaction mix is then cooled down to a temperature of 42 degrees C. to allow primer annealing. This temperature is also the optimal reaction temperature for the combined activities of T7 RNA polymerase and Reverse Transcriptase (RT), which includes an endogenous RNase H activity or is alternatively provided by another reagent. The temperature is kept at 42 degrees C. throughout the following isothermal amplification reaction. The denaturation step, which precedes the amplification cycle, however forces the user to add the enzyme to the test tube after the cool down period in order to avoid inactivation of the enzymes. Therefore, the denaturation step needs to be performed separately from the amplification step.
In accordance with present practice, after adding the test or control sample or both to the amplification reagent mix (typically containing the nucleotides and the primers), the test tube is subject to temperatures xe2x89xa765 degrees C. and then cooled down to the amplification temperature of 42 degrees C. The enzyme is then added manually to start the amplification reaction. This step typically requires the opening of the amplification tube. The opening of the amplification tube to add the enzyme or the subsequent addition of an enzyme to an open tube is not only inconvenient, it also increases the contamination risk.
An alternative approach to amplification of a DNA sample is described in Corbett et al., U.S. Pat. No. 5,270,183. In this technique, a reaction mixture is injected into a stream of carrier fluid. The carrier fluid then passes through a plurality of temperature zones in which the polymerase chain reactions take place. The temperature of the different zones and the time elapsed for the carrier fluid to traverse the temperature zones is controlled such that three events occur: denaturation of the DNA strands, annealing of oligonucleotide primers to complementary sequences in the DNA, and synthesis of the new DNA strands. A tube and associated temperature zones and pump means are provided to carry out the ""183 patent process.
The present invention avoids the inconvenience and contamination risk described above by providing a novel test device in the form of a strip that includes a dual chamber or xe2x80x9cbinaryxe2x80x9d reaction vessel, and a manner of using the device. The invention achieves the integration of the denaturation step with the amplification step without the need for a manual enzyme addition and without exposing the amplification chamber to the environment. The contamination risks from sample to sample contamination within the processing station are avoided since the amplification reaction chamber is sealed and not opened to introduce the patient sample to the enzyme. Contamination from environmental sources is avoided since the amplification reaction chamber remains sealed. The risk of contamination in nucleic acid amplification reactions is especially critical since large amounts of the amplification product are produced. The present invention provides a reaction chamber design that substantially eliminates these risks.
The preferred test strip embodiment allows the test device to be used in a currently installed instrument base after the performance of the amplification reaction, namely the VIDAS(copyright) instrument manufactured and distributed by the assignee of the present invention, bioMxc3xa9rieux, Inc. Thus, providing test devices in a size and configuration to be readily used in an existing instrument base allows the devices to be commercialized and used with a reduced capital expenditure and without having to develop a new instrument for processing the reaction and detecting the resulting amplicons. It will be apparent, however, from the following detailed description that the invention can be practiced in other configurations from the presently preferred embodiment described in detail herein.
In a preferred form of the invention, a dual chamber reaction vessel is provided which comprises a single or unit dose of reagents for a reaction requiring differential heat and containment features, such as a nucleic acid amplification reaction (for example, TMA reaction) packaged ready for use. The dual chamber reaction vessel is designed as a single use disposable unit. The reaction vessel is preferably integrally molded into a test device, such as a strip, having a set of wash and reagent wells for use in a amplification product detection station. Alternatively, the reaction vessel can be made as a stand alone unit with flange or other suitable structures for being able to be installed in a designated space provided in such a test device.
In the dual chamber reaction vessel, two separate reaction chambers are provided in a preferred form of the invention. The two main reagents for the reaction are stored in a spatially separated fashion. One chamber has the heat stable sample/amplification reagent (containing primers, nucleotides, and other necessary salts and buffer components in a reaction solution), and the other chamber contains the heat labile enzymatic reagents, e.g., T7 and RT. Alternatively, the heat labile enzymatic reagents may be stored in an intermediate chamber or well in fluid communication with the second chamber, such that a reaction solution from the first chamber flows through the intermediate chamber en route to the second chamber.
A fluid channel extending from the first chamber to the second chamber links the two chambers to each other. A means is provided for controlling or allowing the flow of fluid through the fluid channel from the first chamber to the second chamber. Various fluid flow control means are contemplated, such as providing a valve in the fluid channel, as described in the application Ser. No. 09/053,823 filed Apr. 2, 1998, now U.S. Pat. Nos. 5,989,499 and 5,786,182. Several different valve embodiments are described therein.
In use, the fluid sample is introduced into the first chamber and the first chamber is heated to a denaturation temperature (e.g., 95 degrees C.). After the amplification reagents in the first chamber have reacted with the fluid sample and the denaturation process has been completed, the first chamber is cooled to 42 degrees C. for primer annealing. The two chambers of the reaction vessel are not in fluid communication with each other prior to completion of the denaturation and cooling step. After these steps are complete, the means for controlling the flow of fluid is operated to allow the reaction solution to pass through the fluid channel from the first chamber to the second chamber. For example, the valve in the fluid channel is opened and the fluid sample is directed into the second chamber either by pressure or vacuum techniques. The reaction solution is then brought into contact with the amplification enzyme(s) (e.g., T7 and/or RT) and the enzymatic amplification process proceeds in the second chamber at 42 degrees C.
In a preferred embodiment, after completion of the amplification reaction, an SPR(copyright) (a fluid transfer, pipette-like device that functions as solid phase receptacle) is introduced into the second chamber. The test strip contains a plurality of wells arranged in an array. Hybridization, washing and optical analysis then proceeds in the wells in accordance with well known techniques in order to detect the amplification products. Such processes may occur in the adjacent wells of a test strip embodiment of the dual chamber reaction vessel automatically in an automated instrument, such as the VIDAS(copyright) instrument of bioMxc3xa9rieux, Inc.
Thus, a preferred form the present invention comprises a test strip for performing a nucleic acid amplification reaction. The test strip includes an integral, elongate housing formed in a strip having a first end and a second end, with the housing having a portion thereof defining a plurality of wells arranged in a linear array disposed in the strip between the first and second ends. The wells will be typically used for hybridization, washing or other detection and decontamination steps after the nucleic acid amplification reaction has been completed.
The test strip housing includes a dual chamber reaction vessel for a nucleic acid amplification reaction to be carried out therein, positioned adjacent to the first end of the test strip. The dual chamber reaction vessel comprises (1) a first chamber in which a first portion of a nucleic acid amplification reaction is performed (e.g., denaturation and primer annealing), (2) a second chamber in which a second portion of a nucleic acid amplification reaction is performed, (e.g., amplification of nucleotides by an amplification enzyme), and (3) an openable connecting conduit linking the first chamber to the second chamber.
In manufacture of the test strip, a first reagent (e.g., primers and/or nucleotides) is inserted into the first chamber for the first portion of the nucleic acid amplification reaction, and a second reagent (e.g., an amplification enzyme) is either inserted into or placed in fluid communication with the second chamber. After the reagents are loaded into the two chambers of the dual chamber reaction vessel (and any required reagent(s) inserted into the remaining wells of the test strip), a membrane is affixed to the test strip housing. The membrane is applied to the housing to cover the first and second chambers and the wells. Separate reagent loading steps and covering may be provided. Such other reagents can be used in other reaction steps for analysis of the test sample.
In order to prolong the shelf life of the test strips and reagents contained therein, a desiccant may be placed into the test strip, preferreably in communication with either the first or second chambers of the dual chamber reaction vessel. Alternatively, the desiccant may be molded into the material forming the test strip housing.
In order to provide a means for decontaminating the test strip before removing it from the instrument, a decontamination solution or bleach wash is provided in one of the sealed wells.